Overview
Pathogenic fungi occur world wide and are major agricultural and health pests. Fungal infections in humans range from superficial and cutaneous to deeply invasive and disseminated. Treatment of fungal infections has lagged behind bacterial chemotherapy. There are substantially fewer antifungal drugs than antibacterial drugs.
In the last two decades, nearly 12 million people have died of acquired immunodeficiency syndrome (AIDS) after being infected with HIV, the human-immunodeficiency virus. AIDS is defined by the occurence of at least one of more than two dozen opportunistic infections. Fungal opportunistic infections such as candidiasis, cryptococcosis, and histoplasmosis, occur frequently in patients with AIDS. Among the opportunistic infections, fungal infections caused by Pneumocystis, Candida, Cryptococcus, or Histoplasma were the first to occur in more than 50% of persons with AIDS; and at time of death, nearly 85% of decedents had a fungal infection (Jones, et al., 1999, MMWR 48: 1-22).
Cryptococcus neoformans is an opportunistic yeast pathogen that infects individuals with a compromised immune system, such as those with AIDS or those undergoing cancer chemotherapy (Kwon-Chung and Bennett, 1992, in Medical Mycology, pp. 397-446; Mitchell and Perfect, 1995, Clin. Microbiol. Rev. 8: 515-548; Sugar, 1991, Mycopathologia 114: 153-157). Its normal habitat is worldwide in pigeon droppings, and one variety is associated with eucalyptus trees (Ellis and Pfeiffer, 1992, Eur. J. Epidemiol. 8: 321-325; Levitz, 1991, Reviews of Infect. Dis. 13: 1163-1169; Pfeiffer and Ellis, 1991, J. Infect. Dis. 163: 929-930). The most common infection caused by this yeast is a lethal meningitis (Chuck and Sande, 1989, N. Engl. J. Med. 321: 794-799; Kovacs, et al., 1985, Ann. Intern. Med. 103: 533-538; Rozenbaum, et al., 1994, Clin. Infect. Dis. 18: 369-380; Stanseld, 1993, Semin. Respir. Infect. 8: 116-123; Zuger, et al., 1986, Ann. Intern. Med. 104: 234-240). Infection with C. neoformans begins with inhalation of infectious particles, and is first established in the lungs. Cryptococcosis is one of the defining diseases associated with AIDS, and nearly 10% of all AIDS patients have cryptococcosis.
Treatments for fungal infections include azole antifungal drugs, such as clotrimazole, ketaconazole, fluconazole, and itraconazole (for a review, see Graybill, 1996, Clin. Infect. Dis. 22: S166-S178). The azoles act by inhibiting an enzyme, lanosterol demethylase, that participates in the synthesis of ergosterol, an essential component of fungal membranes. Other commonly used drugs include flucytosine and amphotericin B. Amphotericin B is one of the most effective antifungal drugs but must be given intravenously and causes serious side effects. Resistance to antifungals has become more apparent in recent years and may worsen with the increase in prophylatic therapy.
There is intense interest in identifying new drugs with different modes of action against fungal infections. The current repertoire of antifungals has limitations such as insufficient efficacy, the need for intravenous administration, serious side effects, or the appearance of resistant fungal strains. Importantly, most of the current treatments are fungistatic, that is, they inhibit fungal growth but do not cause outright death. Subsequent clearing of these inhibited fungi is inadequate in patients with defective immune systems. Thus, it is imperative to identify fungicidal agents and, if possible, their cellular targets that, when impaired, lead to fungal cell death.
Fungi and Antifungal Agents
Fungi are eukaryotic cells that may reproduce sexually or asexually and may be biphasic, with one form in nature and a different form in the infected host. Fungal diseases are referred to as mycoses. Some mycoses are endemic, i.e. infection is acquired in the geographic area that is the natural habitat of that fungus. These endemic mycoses are usually self-limited and minimally symptomatic. Some mycoses are chiefly opportunistic, occurring in immunocompromised patients such as organ transplant patients, cancer patients undergoing chemotherapy, burn patients, AIDS patients, or patients with diabetic ketoacidosis.
Fungal infections are becoming a major health concern for a number of reasons, including the limited number of antifungal agents available, the increasing incidence of species resistant to older antifungal agents, and the growing population of immunocompromised patients at risk for opportunistic fungal infections. The incidence of systemic fungal infections increased 600% in teaching hospitals and 220% in non-teaching hospitals during the 1980's. The most common clinical isolate is Candida albicans (comprising about 19% of all isolates). In one study, nearly 40% of all deaths from hospital-acquired infections were due to fungi. (Sternberg, 1994, Science 266: 1632-1634).
Neutropenic patients (due to, e.g., chemotherapy, immunosuppressive therapy, infection, including AIDS, or an otherwise dysfunctional immune system) are predisposed to the development of invasive fungal infections, most commonly including Candida species and Aspergillus species, and, on occasion, Fusarium, Trichosporon and Dreschlera. Cryptoccocus infection is also common in patients on immunosuppressive agents.
The majority of known antifungal agents fall into one of three main groups. The major group includes polyene derivatives, including amphotericin B and the structurally related compounds nystatin and pimaricin, which are only administered intravenously. These are broad-spectrum antifungals that bind to ergosterol, a component of fungal cell membranes, and thereby disrupt the membranes, leading to cell death. Amphotericin B is usually effective for systemic mycoses, but its administration is limited by toxic effects that include fever and kidney damage, and other accompanying side effects such as anemia, low blood pressure, headache, nausea, vomiting and phlebitis. The unrelated antifungal agent flucytosine (5-fluorocytosine), an orally absorbed drug, is frequently used as an adjunct to amphotericin B treatment for some forms of candidiasis and cryptococcal meningitis. Its adverse effects include bone marrow depression with leukopenia and thrombocytopenia.
The second major group of antifungal agents includes azole derivatives which impair synthesis of ergosterol and lead to accumulation of metabolites that disrupt the function of fungal membrane-bound enzyme systems (e.g., cytochrome P450) and inhibit fungal growth. Significant inhibition of mammalian P450 results in important drug interactions. This group of agents includes ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, terconazole, fluconazole and itraconazole. These agents may be administered to treat systemic mycoses. Ketoconazole, an orally administered imidazole, is used to treat nonmeningeal blastomycosis, histoplasmosis, coccidioidomycosis and paracoccidioidomycosis in non-immunocompromised patients, and is also useful for oral and esophageal candidiasis. Adverse effects include rare drug-induced hepatitis; ketoconazole is also contraindicated in pregnancy. Itraconazole appears to have fewer side effects than ketoconazole and is used for most of the same indications. Fluconazole also has fewer side effects than ketoconazole and is used for oral and esophageal candidiasis and cryptococcal meningitis. Miconazole is a parenteral imidazole with efficacy in coccidioidomycosis and several other mycoses, but has side effects including hyperlipidemia and hyponatremia.
The third major group of antifungal agents includes allylamnines-thiocarbamates, which are generally used to treat skin infections. This group includes tolnaftate and naftifine.
Another antifungal agent is griseoflulvin, a fungistatic agent which is administered orally for fungal infections of skin, hair or nails that do not respond to topical treatment.
Most endemic mycoses are acquired by the respiratory route and are minimally symptomatic; cough, fever, headache, and pleuritic pain may be seen. Occasionally, endemic mycoses may cause progressive pulmonary disease or systemic infection. Histoplasmosis, caused by Histoplasma, is the most common endemic respiratory mycosis in the United States; over 40 million people have been infected. The disease is noncontagious and ordinarily self-limited, but chronic pulmonary infection and disseminated infection may occur. Pulmonary infection rarely requires treatment, but disseminated infection may be treated with amphotericin B. Coccidioidomycosis, caused by Coccidioides, is a noncontagious respiratory mycosis prevalent in the southwest United States. It also is usually self-limited but may lead to chronic pulmonary infection or disseminated infection. Amphotericin B or miconazole may be given for treatment. Blastomycosis, caused by Blastomyces is a noncontagious, subacute or chronic endemic mycosis most commonly seen in the southeast United States. Most pulmonary infections are probably self-limited. Patients with progressive lung disease or disseminated disease, and immunocompromised patients, may be treated systemically with amphotericin B. Paracoccidioidomycosis, caused by Paracoccidioides, is a noncontagious respiratory mycosis that is the most common systemic mycosis in South America. It may be acute and self-limited or may produce progressive pulmonary disease or extrapulmonary dissemination. Disseminated disease is generally fatal in the absence of therapy. Sulfonamides may be used but have a low success rate. Amphotericin B produces a higher response rate but relapses may still occur.
Cryptococcosis is a noncontagious, often opportunistic mycosis. It is characterized by respiratory involvement or hematogenous dissemination, often with meningitis. A major etiologic agent is C. neoformans. Most pulmonary infections are probably overlooked, but cryptococcal meningitis, which accounts for 90% of reported disease, is dramatic and seldom overlooked. Cryptococcosis is a particular problem in immunocompromised patients; cryptococcal meningitis occurs in 7 to 10% of AIDS patients. The principal symptom of meningitis is headache; associated findings include mental changes, ocular symptoms, hearing deficits, nausea, vomiting, and seizures. Without treatment, 80% of patients die within two years. In meningitis, cryptococci can be observed in India ink preparations of cerebrospinal fluid sediment, and can be cultured from the cerebrospinal fluid. Treatment is generally with fluconazole or the combination of amphotericin B and flucytosine, although amphotericin B does not cross the blood brain barrier.
Aspergillosis is a term that encompasses a variety of disease processes caused by Aspergillus species. Aspergillus species are ubiquitous; their spores are constantly being inhaled. Of the more than 300 species known, only a few are ordinarily pathogenic for man: A. fumigatus, A. flavus, A. niger, A. nidulans, A. terreus, A. sydowi, A. flavatus, and A. glaucus. Aspergillosis is increasing in prevalence and is particularly a problem among patients with chronic respiratory disease or immunocompromised patients. Among immunocompromised patients, aspergillosis is second only to candidiasis as the most common opportunistic mycosis and accounts for about 15% of the systemic mycoses in this group. Opportunistic pulmonary aspergillosis is characterized by widespread bronchial erosion and ulceration, followed by invasion of the pulmonary vessels, with thrombosis, embolization and infarction. Clinically, infection manifests as a necrotizing patchy bronchopneumonia, sometimes with hemorrhagic pulmonary infarction. In about 40% of cases, there is hematogenous spread to other sites. Aspergillosis is also a rare but devastating complication of burn wounds; amputation is often required for cure. Invasive aspergillosis is commonly fatal, so aggressive diagnosis and treatment is required. Blood, urine and cerebrospinal fluid cultures are rarely positive, but fungi can be seen in smears and biopsies. Amphotericin B can be given for treatment.
Dermatophytosis is a chronic fungal infection of the skin, hair or nails by dermatophytes, which include members of the species Trichophyton, Microsporum and Epidermophyton. Infection of the foot (tinea pedis), scalp (tinea capitis) are most common, although widespread infection on non-hair-bearing skin (tinea corporis) also occurs. Clinical manifestations vary and may present on the skin as fissuring or lesions with scaling, vesicles or pustules (and alopecia on the scalp), or on the nails as discolored or chalky, crumbling nails. Both topical and systemic therapies may be used to treat dermatophyte infection, including topically administered imidazoles and triazoles (such as itraconazole, miconazole, ketoconzaole and econzaole), haloprogin, undecylic acid, ciclopirox olamine, tolnaftate and terbinafine.
Fusarium species can cause localized or hematogenously disseminated infection (fusariosis), most frequently in patients who have a hemopoietic malignancy and neutropenia. Abrupt onset of fever, sometimes with myalgia, is followed in the majority of cases by distinctive skin lesions resembling ecthyrna gangrenosum. Infection can be treated with amphotericin B but recovery depends ultimately on alleviation of neutropenia. Mortality typically exceeds 90%.
Mucormycosis is an acute suppurative opportunistic mycosis that produces rhinocerebral, pulmonary or disseminated disease in immuno-compromised patients, and local or disseminated disease in patients with burns or open wounds. Infection is caused by fungi in the class Zygomycetes, and include Basidiobolus, Conidiobolus, Rhizopus, Mucor, Absidia, Mortierella, Cunninghamella, and Saksenaea Rhinocerebral mucormycosis accounts for about half of all cases of mucormycosis. It is one of the most rapidly fatal fungal diseases, with death occurring within 2-10 days in untreated patients. Early clinical signs include nasal stuffiness, bloody nasal discharge, facial swelling and facial pain. The infection then spreads to the eyes, cranial nerves and brain. Pulmonary mucormycosis is nearly as common as rhinocerebral disease and manifests with the same necrotizing and infarction as aspergillosis. Fungi are virtually never seen or cultured from blood, sputum or cerebrospinal fluid. Disseminated mucormycosis may follow pulmonary or burn wound infection. Treatment is with amphotericin B.
Candidiasis is a general term for a variety of local and systemic processes caused by colonization or infection of the host by species of the yeast Candida. Candidiasis occurs worldwide; superficial infections of the skin, mouth and other mucus membranes are universal. Invasive systemic disease has become a problem due to the use of high doses of antibiotics that destroy normal bacterial flora, immunosuppressive agents, and agents toxic to bone marrow, e.g., during cancer therapy. Neutropenia is a major risk factor for Candida dissemination. Candidiasis is also seen among immunocompromised individuals such as AIDS patients, organ transplant patients, patients receiving parenteral nutrition, and cancer patients undergoing radiation treatment and chemotherapy. It is the most common opportunistic mycosis in the world. The most common etiologic agent is Candida albicans. Other infectious species include C. tropicalis, C. parapsilosis, C. stellatoidea, C. kusei, C. parakwsei, C. lusitaniae, C. pseudotropicalis, C. guilliermondi and C. glabrata. Candida albicans is normally found in the mouth, throat, gastrointestinal tract and vagina of humans. Non-albicans species frequently colonize skin. Candida species occur in two forms that are not temperature- or host-dependent. The usual colonizing forms are yeasts that may assume a pseudomycelial configuration, especially during tissue invasion. Pseudomyceliae result from the sequential budding of yeasts into branching chains of elongated organisms.
Candida albicans contains cell wall mannoproteins that appear to be responsible for attachment of the yeast cells to specific host tissues. It has been reported that the mannan portion, rather than the protein portion, of the mannoproteins is responsible for adherence of finmgal cells to spleen and lymph node tissues in mice. (Kanbe, et al., 1993, Infection Immunity 61: 2578-2584).
C. albicans also binds avidly to extracellular matrix (ECM) proteins such as fibronectin, laminin, and types I and IV collagen, all of which contain heparin-binding domains. This suggests C. albicans may express a heparin-like surface molecule. Adherence of C. albicans to the ECM may be important in the pathogenesis of disseminated candidiasis. It has been demonstrated that heparin, heparan sulfate and dextran sulfate glycosaminoglycans (GAGs) inhibit adherence of C. albicans to ECM and ECM proteins, possibly by a mechanism involving binding of GAGs to ECM proteins, thus masking these selective ligands. (Klotz, et al, 1992, FEMS Microbiology Letters 78: 205-208).
Clinically, candidiasis manifests as superficial mucocutaneous infections, chronic mucocutaneous candidiasis, or systemic infection. Superficial mucocutaneous infections can occur in any area of skin or mucus membrane. Thrush, commonly seen in AIDS patients, is characterized by a patchy or continuous, creamy to gray pseudomembrane that covers the tongue, mouth, or other oropharyngeal surfaces and may be accompanied by ulceration and necrosis. Laryngeal involvement results in hoarseness. Esophagitis is often an extension of oropharyngeal disease and may manifest with symptoms of retrosternal pain and dysphagia. Intestinal candidiasis is commonly asymptomatic, but is a major source of hematogenous invasion in immunocompromised individuals. Intertrigo involves the axillae, groins, inframammary folds, and other warm, moist areas, and may manifest as red, oozing or dry, scaly lesions. Infections may occur in other areas, including perianal and genital areas. Paronychia, infection of the nails, often follows chronic exposure of the hands or feet to moisture. Some patients with limited T-cell immunodeficiency develop chronic mucocutaneous candidiasis. These patients suffer from persistent superficial Candida infection of the skin, scalp, nails and mucus membranes.
Most cases of systemic candidiasis are caused by Candida albicans and C. tropicalis, and increasingly, C. glabrata. Clinical manifestations of Candida infection appear mainly in the eyes, kidneys and skin. In the eyes, there may be single or multiple raised, white, fluffy chorioretinal lesions. These lesions are a potential cause of blindness. Involvement of the kidneys includes diffuse abscesses, capillary necrosis and obstruction of the ureters. Infection may result in progressive renal insufficiency. Systemic Candida infection can also manifest as maculonodular skin lesions surrounded by a reddened area; these lesions have an appearance similar to acne but are a major clue to a potentially lethal disease. Other manifestations of systemic candidiasis may include osteomyelitis, arthritis, meningitis, and abscesses in the brain, heart, liver, spleen and thyroid. Involvement of the lungs is also common, but pulmonary lesions are usually too small to be seen on chest X-ray. Finally, Candida endocarditis can occur in patients receiving prolonged intravenous therapy or cardiac valve implants, or in intravenous drug abusers. Fungal lesions appear on the valves, and can embolize and occlude large blood vessels.
Superficial infections are diagnosed by microscopic examination of scrapings or swabs of infected lesions in the presence of 10% potassium hydroxide. Candida organisms can also be seen on gram stain. Endocarditis is diagnosed by blood cultures or demonstration of bulky valvular lesions on echocardiography. Systemic candidiasis may be difficult to diagnose because the presence of heavy colonization at the usual sites of infection indicates, but does not prove, that dissemination has occurred. The most reliable evidence of systemic candidiasis is biopsy demonstration of tissue invasion or recovery of yeast from fluid in a closed body cavity, such as cerebral spinal fluid, pleural or peritoneal fluid. Similarly, positive blood or urine or sputum cultures may indicate invasive disease or simply localized disease around indwelling devices, e.g., catheters or intravenous lines.
Mucocutaneous infections may be treated with topical preparations of nystatin, amphotericin B, clotximazole, miconazole, haloprogin or gentian violet. Oropharyngeal or esophageal candidiasis can be treated with systemic agents such as ketoconazole or fluconazole. Chronic mucocutaneous candidiasis syndrome may respond to topical or systemic therapeutic agents such as amphotericin B or ketoconazole, but often relapses when medication is discontinued. Cystitis may be treated with amphotericin B bladder rinses, or a brief low-dose intravenous course of amphotericin B with or without oral flucytosine. Endocarditis is essentially incurable without valve replacement, accompanied by a 6 to 10 week course of amphotericin B and flucytosine. Even with therapy, however, complete cure of endocarditis is not always possible.
The mortality rate from systemic candidiasis is about 50%. Systemic candidiasis may be treated with fluconazole, a fungistatic agent, or amphotericin B, a fungicidal agent although systemic use of the latter is limited by its toxicity. Both drugs have substantial adverse reactions when used in combination with cyclosporine A, which itself can be nephrotoxic. The removal of precipitating factors such as intravenous lines or catheters is also important for controlling infection. Flucytosine therapy can be added to the amphotericin B therapy for treatment of systemic candidiasis, especially in patients that are not imnmunocompromised. In immunocompromised patients, however, these infections are problematic and resist effective treatment. Mortality with systemic candidiasis can be over 90% in such patients. Furthermore, chronic mucocutaneous candidiasis and candidal endocarditis often show evidence of disease after having been declared cured.
Infection of the cornea and conjunctiva, including keratoconjunctivitis, can result from infection by amoeba, viruses, fungi and bacteria. Debilitated patients can develop keratitis from fungi such as Candida or Fusarium which is often associated with corneal ulceration and can lead to scarring with severe visual loss.
Amiodarone
Amiodarone is a Class III antiarrhythnic drug (Amiodarone in Physicians GenRx, 1996, BeDell, et. al, eds., Mosby-Year Book, Inc., St. Louis, Mo.; Amiodarone in Drug Information for the HealthCare Profession, 1997, USP DI, Twinbrook Parkway, Md; pp. 80-83). It is administered orally in 200 mg tablets and is sold under the name Cordarone.RTM. in the United States and several other brand names outside the U.S. Amiodarone prolongs the repolarization phase of the cardiac action potential. Amiodarone has beneficial effects in the treatment of patients with ventricular arrhythnias after myocardial infarction. Amiodarone therapy for arrhythmia requires prolonged treatment on the order of months to years. However, amiodarone dosed for this use has several unwanted side-effects, including thyroid dysfunction, hepatitis, impaired vision, photosensitivity of the skin, and pneumonitis. Amiodarone therapy for arrhythmia requires prolonged treatment on the order of months to years.
In animals, amiodarone is effective in the prevention or suppression of experimentally induced arrhythmias. The antiarrhythmic effect of amiodarone may be due to at least two major properties: 1) a prolongation of the myocardial cell-action potential duration and refractory period; and 2) noncompetitive alpha- and beta-adrenergic inhibition. Amiodarone prolongs the duration of the action potential of all cardiac fibers while causing minimal reduction of dV/dt. The refractory period is prolonged in all cardiac tissues.
Following oral administration in man, aniodarone is slowly and variably absorbed. The bioavailability of amiodarone is approximately 50% but has varied between 35 and 65% in various studies. Maximum plasma concentrations are attained 3 to 7 hours after a single dose. Despite this, the onset of Amiodarone's antiarrhythmic effect may take several days to 1 to 3 weeks to occur, even when administered with a loading dose. Plasma concentrations with chronic dosing at 100 to 600 mg/day are approximately dose proportional, with a mean 0.5 mg/L increase for each 100 mg/day administered. However, considerable individual variability occurs.
Amiodarone has a very large but variable volume of distribution, averaging about 60 L/kg because of extensive accumulation in various sites, especially adipose tissue and highly perfused organs, such as the liver, lung, and spleen. One major metabolite of amiodarone, desethylamiodarone, has been identified in man; it accumulates to an even greater extent in almost all tissues. The pharmacological activity of this metabolite, however, is not known. During chronic treatment, the plasma ratio of metabolite to parent compound is approximately one. The main route of amiodarone elimination is via hepatic excretion into bile.
There may be significant adverse side effects with the use of amiodarone. These include neurotoxicity, photosensitivity, and pulmonary fibrosis or interstitial pneumonitis/alveolitis. Ataxia is the most common symptom, occurring in 20-40% of patients, especially during administration of loading doses. It may occur within 1 week to several months after initiation of therapy and may persist for more than a year after withdrawal. Photosensitivity may require ultraviolet-A sun-block, such as zinc or titanium oxide and protective clothing. Pulmonary fibrosis or interstitial pneumonitis/alveolitis is clinically significant in up to 10 to 15% of patients, but abnormal diffusion capacity occurs in a much higher percentage of patients, more frequently with doses of 400 mg/day and after several months of treatment. It is usually reversible after withdrawal of amiodarone but is fatal in about 10% of cases, especially when not diagnosed promptly.
Incidence of adverse side effects is generally related to dose and duration of therapy. Adverse side effects may occur even at therapeutic plasma amiodarone concentrations, but are more common at concentrations over 2.5 .mu.g per ml and with continuous treatment for longer than 6 months.
Amiodarone has also been found to affect mammalian G protein activity by Hageluken, et al., 1995, Mol. Pharmacol. 47: 234-240 who reported that amiodarone increased high affinity GTP hydrolysis with an EC50 of 7.5 .mu.M and stimulated binding of guanosine-5.mu.-O-(3-thio)triphosphate to, and incorporation of GTP azidoanilide into Gi protein subunits in HL-60 membranes. These authors also found that amiodarone increased the cytosolic Ca.sup.2+ concentration in HL-60 cells in the presence but not in the absence of extracellular Ca.sup.2+. In vitro, amiodarone activated the GTPase of reconstituted Gi/Go proteins and Gi2 with EC50 values of 20 .mu.M and 50 .mu.M, respectively. These authors concluded that amiodarone is a direct activator of Gi and Go proteins and that amiodarone activates nonselective cation channels in HL-60 cells via Gi proteins.
There continues to exist a need in the art for new products and methods for their use as antifungal agents. In particular, effective antifungal therapy for systemic mycoses is limited. Products and methods responsive to this need would ideally involve substantially non-toxic compounds available in large quantities. Ideal compounds would have a rapid effect and a broad spectrum of fungicidal or fungistatic activity against a variety of different fungal species when administered or applied as the sole antifungal agent. Ideal compounds would also be useful in combinative therapies with other antifungal agents, particularly where these activities would reduce the amount of antifungal agent required for therapeutic effectiveness, enhance the effect of such agents, or limit potential toxic responses and high cost of treatment. Particularly advantageous would be compounds that are orally available and active for administration of antifungal agents.